Download DNA - morescience

Chapter 16.
DNA
The Genetic Material
Replication
Scientific History
 The march to understanding that DNA is
the genetic material
T.H. Morgan (1908)
 Frederick Griffith (1928)
 Avery, McCarty & MacLeod (1944)
 Hershey & Chase (1952)
 Watson & Crick (1953)
 Meselson & Stahl (1958)

1908 | 1933
Genes are on chromosomes
 T.H. Morgan



working with Drosophila (fruit flies)
genes are on chromosomes
but is it the protein or the DNA of the
chromosomes that are the genes?
 through 1940 proteins
were thought to be
genetic material… Why?
What’s so impressive
about proteins?!
Protein
20 different amino acids
copious amounts and variations of proteins
Found everywhere
vs
DNA
4 different bases (A, T, G, C)
limited amounts in each cell
found mostly in the nucleus
The “Transforming Factor”
 Frederick Griffith




1918 – the world was struck
by a pandemic (cross continents)
an influenza that turned to
pneumonia, killing an estimated
30 – 50 million people
was working to find cure for pneumonia
Streptococcus pneumonia bacteria
He “stumbled upon” a substance that was
passed from dead bacteria to live bacteria
= “Transforming Factor”
1928
In Frederick Griffith’s 1920’s experiments:
1. What happened to the mice he injected with the
R-Strain bacteria?
2. What happened to the mice he injected with the
S-Strain bacteria that causes pneumonia?
3. What happened to the mice he injected with the
Heat-killed S-Strain bacteria that causes
pneumonia?
4. What happened to the mice he injected with the
R-Strain bacteria AND the heat-killed S-Strain
bacteria?
Frederick Griffith’s conclusions:
1. The living bacteria recovered from the
dead mice were the S (smooth) Strain.
“Something” must have “Transformed”
the R-Strain to become smooth.
2. He proposed that when the smooth
bacteria were killed…
“something” from inside the S-Strain
bacteria must have been released into the
media upon the death of the bacteria.
3. Whatever that transforming factor was
must have instructed the non-pathogenic
R-Strain bacteria to become pathogenic SStrain bacteria.
The “Transforming Factor”
live pathogenic
strain of bacteria
A.
mice die
live non-pathogenic heat-killed
strain of bacteria
pathogenic bacteria
B.
C.
mice live
mice live
mix heat-killed
pathogenic &
non-pathogenic
bacteria
D.
mice die
Transformation?
something in heat-killed bacteria could still transmit
disease-causing properties
1944
DNA is the “Transforming Factor”
 Avery, McCarty & MacLeod

purified both DNA & proteins from
Streptococcus pneumonia bacteria
 which will transform non-pathogenic bacteria?

injected protein into bacteria
 no effect

injected DNA into bacteria
 transformed harmless bacteria
into virulent bacteria
What’s the
conclusion?
What was Oswald Avery’s conclusion when he
used enzymes to break down specific bacterial
components in this experiment?
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as
Benjamin Cummings.
Conclusion – Griffith’s “Transforming Factor” was DNA, not RNA or protein
Confirmation of DNA
 Hershey & Chase
1952 | 1969
classic “blender” experiment
 worked with bacteriophage

 viruses that infect bacteria

Why use
Sulfur
vs.
Phosphorus?

grew phage viruses in 2 media,
radioactively labeled with either

35S
in their proteins
 32P in their DNA
infected bacteria with
labeled phages
Confirmed DNA is “transforming factor”
Protein coat labeled
with 35S
Hershey
& Chase
DNA labeled with 32P
T2 bacteriophages
are labeled with
radioactive isotopes
S vs. P
bacteriophages infect
bacterial cells
Watch video clip
03 Hershey and Chase
bacterial cells are agitated
to remove viral protein coats
Which
radioactive
marker is found
inside the cell?
Which molecule
carries viral
genetic info?
35S
radioactivity
found in the medium
32P
radioactivity found
in the bacterial cells
Blender experiment
 Radioactive phage & bacteria in blender

35S
phage
 radioactive proteins stayed in supernatant
 therefore protein did NOT enter bacteria
 32
P phage
 radioactive DNA stayed in pellet
 therefore DNA did enter bacteria

Confirmed DNA is “transforming factor”
Taaa-Daaa!
Experiments Overview
Griffith’s Experiments (Streptococcus pneumoniae)
Experiment
Results
Conclusion
1
R-Strain bacteria
S-Strain bacteria
Mouse lived
Mouse died
Nonvirulent strain of bacteria
Virulent/deadly strain
2
Heat-killed S-Strain
Mouse lived
Destroyed deadly bacteria
3
Heat-killed S-Strain
+ R-Strain
Mouse died
Some how the killed bacteria was able to
pass “something” to the R-strain
That Transformed it to become deadly
Avery’s Experiments (enzymes & bacteria)
Experiments
Results
Conclusion
Enzymes to break down
proteins, carbohydrates,
lipids, RNA, & finally DNA
Deadly in spite of all DNA is the transforming molecule that
enzymes except one made Griffith’s R-Strain bacteria turn
that broke apart DNA
into an S-Strain bacteria
Hershey & Chase Experiments (bacteria & a virus called bacteriophage)
Experiments
Radioactive Protein
Radioactive DNA
Results
Conclusion
New phages = no radioactivity Protein does not make new phages
DNA makes new phages
New phages = radioactive
Structure of DNA
1953 James D. Watson/Francis H. Crick
proposed the Double Helix Model
based on two sources of information:
1. X-ray crystallography studies by:
Rosalind Franklin
1920 - 1958
Maurice Wilkins
1916 - 2004
2. Chargaff Base-Pair Ruling
15
1947
Chargaff
 DNA composition: “Chargaff’s rules”
varies from species to species
 all 4 bases not in equal quantity
 bases present in characteristic ratio

 humans:
A = 30.9%
T = 29.4%
G = 19.9%
C = 19.8%
What do
you notice?!
Base pairing in DNA
 Purines


adenine (A)
guanine (G)
 Pyrimidines


thymine (T)
cytosine (C)
 Pairing
A:T
C G

Memory technique: All Girls are Pure, Cut The Pie
Critical Thinking: How do the number of purines compare to the number of pyrimidines?
Double helix structure of DNA
the structure of DNA suggested a mechanism for
how DNA is copied by the cell
Directionality of DNA
 You need to
PO4
nucleotide
number the
carbons!

it matters!
N base
5
5 CH2
1
4
3
2
This
will be
IMPORTANT!!
O
4
deoxyribose
3
OH
2
1
The DNA backbone
 Putting the DNA
backbone together

refer to the 3 and 5
ends of the DNA
 the last trailing carbon
I mean it…
This will be
IMPORTANT!!
5
PO4
base
CH2
O
C
O
–O P O
O
CH2
base
O
OH
3
Anti-parallel strands
 Phosphate to sugar bond
involves carbons in 3 & 5
positions
DNA molecule has
“direction”
 complementary strand
runs in opposite direction

“It has not escaped our notice that the specific pairing
we have postulated immediately suggests a possible
copying mechanism for the genetic material.”
Watson & Crick
Bonding in DNA
5’
hydrogen
bonds
3’
phosphodiester
bonds
3’
glycosidic
bonds
5’
….strong or weak bonds?
How do the bonds fit the mechanism for copying DNA?
Models of DNA Replication
 Alternative models

so how is DNA copied?
verify through
experiments…
Semi-conservative replication
1958
 Meselson & Stahl


label nucleotides of “parent” DNA strands with
heavy nitrogen = 15N
label new nucleotides with lighter isotope = 14N
“The Most Beautiful Experiment in Biology”
parent
make predictions…
replication
Semi-conservative replication
 Make predictions…
 15


N strands replicated in 14N medium
1st round of replication?
2nd round?
1958
Copying DNA
 Replication of DNA

base pairing allows
each strand to serve
as a pattern for a
new strand
DNA Replication
let’s meet
the team…
 Large team of enzymes coordinates replication
What’s it really look like?
How many replication bubbles
do you see?
1
2
3
4
Replication: 1st step
 Unwind DNA

helicase enzyme
 unwinds part of DNA helix (replication fork)
 stabilized by single-stranded binding proteins
single-stranded binding proteins
Replication Fork
4th – DNA
Polymerase I
Replaces the
RNA primer
with a DNA
nucleotide
Lagging Strand
3rd – DNA
Polymerase III
adds DNA
2nd –
nucleotides
Primase
attaches
a RNA
Primer
Okazaki Fragments
5’
3’
5th – Ligase “glues”
Okazaki fragments together
on the lagging strand.
3’
5’
5’
3’
Leading Strand
5’
3’
direction of replication?
1st – Helicase unzips a
portion of DNA
Single-Stranded
Binding Proteins
Control the unzipping
Leading & Lagging strands
Leading strand
- continuous synthesis
Built across from 3’ end
In the 5’ to 3’ direction
Okazaki
Lagging strand
- Okazaki fragments
- joined by ligase
- “spot welder” enzyme
Replication: 2nd step
 Bring in new nucleotides to
match up to template strands
But… the
Where’s
We’re
missing
ENERGY
forsomething!
the bonding!
What?
Energy of Replication
 Where does the energy for the bonding come
from?
energy
You
remember
ATP!
Is that the
only energy
molecule?
CTP
TTP
GTP
ATP
AMP
CMP
TMP
GMP
ADP
Energy of Replication
 The nucleotides arrive as nucleosides



DNA bases with P–P–P
DNA bases arrive with their own energy source
for bonding
bonded by DNA polymerase III
ATP
GTP
TTP
CTP
5'
Replication
energy
DNA
P III
 Adding bases
can only add
nucleotides across
from the 3 end of a
growing DNA
strand!!
 New strand grows
5'3’

3'
energy
DNA
P III
energy
energy
B.Y.O. ENERGY
3'
leading strand
5'
5'
3'
5'
3'
ligase
energy
3'
lagging strand 5'
3'
leading strand
5'
Modeling Replication
Make a DNA molecule (paper strip)
5’
3’
3’
5’
Then replicate it
Animation Analysis:
1. Red/Orange = ____________
How do you know? ________
2. Grey/White = ____________
How do you know? ________
3. Green = ________________
How do you know? ________
4. Yellow =
How do you know? ________
5. Blue =
How do you know? ________
6. Pink =
How do you know? ________
7. Bonds =
How do you know? ________
8. What breaks off?
How do you know? ________
9. Why? __________________
10. What were the molecules? ___________
11. What do they become? ______________
12. Where is the 5’ and 3’ on the template &
complementary strands?
Regulating Gene Expression
As shown in the diagram, a chromosome in
the nucleus of a eukaryotic cell is comprised
of DNA wound tightly around histone
proteins, packaging the DNA molecule into
highly compact form called chromatin.
This makes DNA inaccessible to enzymes
that would code for the genetic
Information.
This diagram shows acetyl groups
attaching to the histones.
This causes the tight compaction to
unravel, now allowing DNA to be
susceptible to activation (replication or
transcription)
Regulating Gene Expression
A methyl group (CH3) can be attached to a cytosine base on DNA, as
shown here.
When a methyl group is attached to a base, enhancer, promoter, and
activator proteins cannot access the base to build nucleotides
Implications: What would you expect (in terms of gene expression)
for a gene that contains many methyl groups?
The presence of the methyl group would probably block
transcription from occurring, so the gene would not be expressed.
Okazaki fragments
Priming DNA synthesis
 DNA polymerase III
can only extend an
existing DNA molecule

cannot start new one
 cannot place first base

short RNA primer is
built first by primase
 starter sequences
 DNA polymerase III can
now add nucleotides to
RNA primer
Cleaning up primers
DNA polymerase I
removes sections of
RNA primer and
replaces with DNA
nucleotides
Replication bubble
Adds 1000 bases/second!
 Which direction does DNA build?
 List the enzymes & their role
Draw & Label using Word Bank
lagging strand
leading strand
Okazaki fragments
DNA polymerase I
DNA polymerase III (3x)
helicase
primase
ligase
SSBP
3’
5’
5’
3’
5’
3’
5’
3’
Replication fork
direction of replication
Replication enzymes:
Helicase – unzip helix structure
Single Strand Binding Proteins (SSBP) – control unzipping
Primase – lay down RNA Primer
DNA Polymerase III – extends existing DNA beyond the primer
DNA Polymerase I – replaces RNA Primer with DNA
Ligase – “welds”/glues/bonds the Okazaki Fragments
Replication fork
DNA
polymerase I
DNA
polymerase III
lagging strand
Okazaki
fragments
5’
3’
ligase
primase
5’
SSB
3’
DNA
polymerase III
5’
3’
leading strand
direction of replication
3’
5’
helicase
And in the end…
 Ends of
chromosomes
are eroded with
each replication
an issue in
aging?
 ends of
chromosomes
are protected by
telomeres

Telomeres
 Expendable,
non-coding sequences
at ends of DNA


short sequence of
bases repeated 1000s
times
TTAGGG in humans
 Telomerase enzyme in
certain cells


enzyme extends
telomeres
prevalent in cancers
 Why?
DNA polymerases
 DNA polymerase III


1000 bases/second
main DNA building enzyme
 DNA polymerase I

20 bases/second

primer removal, editing, & repair
DNA polymerase III enzyme
RNA is temporary so nature has not invested in a proof-reader for it!
Editing & proofreading DNA
 1000 bases/second =
lots of typos!
 DNA polymerase I

proofreads & corrects
typos

repairs mismatched bases

excises abnormal bases
 repairs damage
throughout life

reduces error rate from
1 in 10,000 to
1 in 100 million bases
UV radiation can cause molecular
lesions, where T’s or C’s form a
covalent bond between themselves
Fast & accurate!
 It takes E. coli <1 hour to copy
5 million base pairs in its single
chromosome

divide to form 2 identical daughter cells
 Human cell copies its 6 billion bases &
divide into daughter cells in only few
hours
remarkably accurate
 only ~1 error per 100 million bases
 ~30 errors per cell cycle

The “Central Dogma”
 flow of gene
 genetic information within a cell
transcription
DNA
replication
RNA
translation
protein
Check for understanding
#1